The position and movement of synaptic vesicles in presynaptic terminals are important for synaptic transmission. However, accurate three-dimensional tracking of single synaptic vesicles in presynaptic terminals has remained a challenge. Using dual-focus imaging optics, we have been able to track quantum dot (Qdot)-labeled single vesicles in3D, with an accuracy of 20-30 nm in 10 Hz imaging. Using different loading protocols, we are able to track the dynamics of synaptic vesicles derived from either the readily releasable pool or total recycling pool. Also two different modes of exocytosis - full-collapse-fusion and kiss-and-run – could be distinguished using the degree of quenching of quantum dot photoluminescence by micromolar trypan blue.

In order to determine the location of releasing position relative the center of the active zone, we localized the centroids of spectrally separable markers, FM 4-64 (presynaptic sites) and PSD 95-GFP (postsynaptic sites) in 3D using the same methods as Qdot loaded vesicles. The vector connecting these centroids approximated the central axis of the synapse, and allowed us to estimate the degree to which fusion occurred at positions away from the center of the active zone. We found that synaptic vesicles undergoing kiss-and-run tend to fuse close to the center of the active zone whereas vesicles undergoing full-collapse fusion tend to fuse all around the synapse. This difference of fusion mode may be related to the difference in the spatial distribution of AMPA and NMDA receptors, implying the relation between fusion mode and synaptic transmission.